caa a22 4500 475746473 CHVBK 20180406123514.0 cr unu---uuuuu 170329e20001201xx s 000 0 eng 10.1007/s002840010163 doi (NATIONALLICENCE)springer-10.1007/s002840010163 Pesticidal and Receptor Binding Properties of Bacillus thuringiensis Cry1Ab and Cry1Ac δ-Endotoxin Mutants to Pectinophora gossypiella and Helicoverpa zea [Elektronische Daten] [Shahid Karim, Donald H. Dean] Bacillus thuringiensis produces several larvicidal crystalline inclusions during sporulation. An understanding of their mechanisms of action is commercially important. In this study, two toxins, Cry1Ab and Cry1Ac, were compared that showed 98% amino acid identity in domain I and II, but differed significantly in domain III. Using site-directed mutagenesis techniques, two conserved loop 2 Arg's (368RR369) of Cry1Ab and Cry1Ac toxins were replaced with Ala (368AR369, 368RA369, 368AA369), Glu (368EE369), Phe (368FF369), His (368HH369), and Lys (368KK369). The effect of these mutants on structural stability, larvicidal potency, receptor binding, and ionic permeability towards two important cotton pests, pink bollworm (Pectinophora gossypiella) and bollworm (Helicoverpa zea) were analyzed. All seven mutants of Cry1Ab, excluding 368AR369, produced a stable protoxin, whereas for Cry1Ac all seven mutants yielded stable protoxin. Results showed that all the stable mutants behaved similarly to the wild type on incubation with trypsin and gut extract of both insect larvae. The Cry1Ab mutants, 368AR369, 368AA369, 368FF369, and 368HH369, lost toxicity; 368EE369 had reduced toxicity; whereas the more conserved change 368KK369 retained the toxicity similar to the wild type towards P. gossypiella. Double mutants of Cry1Ac, 368AA369 and 368FF369, abolished the toxicity. Double mutant 368KK369 of Cry1Ac retained its toxicity against P. gossypiella, whereas single mutants 368AR369, 368RA369, and 368HH369 retained only reduced toxicity. All the mutants of Cry1Ab lost their toxicity against H. zea except 368KK369. In Cry1Ac single mutants, 368AR369 and 368RA369, reduction in the toxicity was observed. A double mutant of Cry1Ac, 368KK369, also retained reduced toxicity. All the other double mutants lost their toxicity. Voltage clamping experiments on H. zea midguts provided an additional evidence about the insecticidal property and inhibition of Isc across the transepithelial membrane of the insect midgut. Springer-Verlag New York Inc., 2000 Karim Shahid Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA, USA aut Dean Donald H. Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA, USA aut Current Microbiology Springer-Verlag 41/6(2000-12-01), 430-440 0343-8651 41:6<430 2000 41 284 https://doi.org/10.1007/s002840010163 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s002840010163 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Karim Shahid Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA, USA aut NATIONALLICENCE 700 1- Dean Donald H. Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA, USA aut NATIONALLICENCE 773 0- Current Microbiology Springer-Verlag 41/6(2000-12-01), 430-440 0343-8651 41:6<430 2000 41 284 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer